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Mutant receptors and their use in a nuclear receptor-based inducible gene expression system

a gene expression system and nuclear receptor technology, applied in the field of biotechnology or genetic engineering, can solve the problems of system limitation, leakage of system, limited use of pr1-a,

Active Publication Date: 2008-06-19
PRECIGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about a new way to control gene expression in cells. It involves using special proteins called nuclear receptors that can be activated or deactivated to turn genes on or off. These receptors have a specific structure that allows them to bind to DNA and control the expression of genes. The patent describes how these receptors can be modified to have a different function and how they can be used to control gene expression in a way that is more precise and flexible. This technology can be useful for studying and manipulating developmental processes and has applications in genetic engineering and biotechnology.

Problems solved by technology

The use of PR1-a may be limited because it often responds to endogenous materials and external factors such as pathogens, UV-B radiation, and pollutants.
However, these systems have limitations due to their effect on expression of non-target genes.
These systems are also leaky.
Unfortunately, the use of such systems is restricted by unstable chemistry of the ligands, i.e. tetracycline and lactose, their toxicity, their natural presence, or the relatively high levels required for induction or repression.
For similar reasons, utility of such systems in animals is limited.
Unfortunately, this system includes immunosuppressants that can have unwanted side effects and therefore, limits its use for various mammalian gene switch applications.
Unfortunately, use of steroidal compounds that activate the receptors for the regulation of gene expression, particularly in plants and mammals, is limited due to their involvement in many other natural biological pathways in such organisms.
Unfortunately, these USP-based systems are constitutive in animal cells and therefore, are not effective for regulating reporter gene expression.
Drawbacks of the above described EcR-based gene regulation systems include a considerable background activity in the absence of ligands and non-applicability of these systems for use in both plants and animals (see U.S. Pat. No. 5,880,333).

Method used

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  • Mutant receptors and their use in a nuclear receptor-based inducible gene expression system
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  • Mutant receptors and their use in a nuclear receptor-based inducible gene expression system

Examples

Experimental program
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Effect test

example 1

[0261]This Example describes the construction of several gene expression cassettes comprising novel substitution mutant Group H nuclear receptor polynucleotides and polypeptides of the invention for use in a nuclear receptor-based inducible gene expression system. Applicants constructed gene expression cassettes based on the spruce budworm Choristoneura fumiferana EcR (“CfEcR). The prepared receptor constructs comprise a ligand binding domain of either an EcR or a chimera of Homo sapiens RXRβ-LmRXR; and a GAL4 DNA binding domain (DBD) or a VP16 transactivation domain (AD). The reporter constructs include the reporter gene luciferase operably linked to a synthetic promoter construct that comprises a GAL4 response element to which the Gal4 DBD binds. Various combinations of these receptor and reporter constructs were cotransfected into mammalian cells as described in Examples 2-5 infra.

[0262]Gene Expression Cassettes: Ecdysone receptor-based gene expression cassettes (switches) were c...

example 2

[0271]This Example describes the identification of ecdysteroid responsive CfEcR ligand binding domain substitution mutants that exhibit increased activity in response to ecdysteroidal ligand. In an effort to identify substitution mutations in the CfEcR that increase ecdysteroidal ligand activity, Applicants mutated amino acid residues predicted to be critical for ecdysteroid binding and created GAL4 / mutantCfEcR-DEF cDNA gene expression cassettes as described in Example 1 above using PCR-mediated site-directed mutagenesis kit. The mutated and the WT cDNAs corresponding to the various switch constructs outlined above in Example were made and tested in GAL4-driven luciferase reporter assays as described below.

[0272]Transfections: DNAs were transfected into mouse NIH3T3 cells (ATCC) as follows. Standard methods for culture and maintenance of the cells were followed. Cells were harvested and plated 96-well plates at 2,500 cells per well, in 50 μL of growth medium containing 10% fetal bov...

example 3

[0298]This Example describes the identification of CfEcR ligand binding domain substitution mutants that are generally ecdysteroid responsive that exhibit increased activity in response to ecdysteroids. In an effort to identify substitution mutations in the CfEcR that increase ecdysteroid activity, Applicants mutated amino acid residues and created GAL4 / mutant CfEcR-DEF cDNA gene expression cassettes as described in Example 1 above using PCR-mediated site-directed mutagenesis kit. The mutated and the WT cDNAs corresponding to the various switch constructs outlined above in Example 1.1 and 1.2 were made and tested in a GAL4-driven luciferase reporter assay as described in Example 2.

[0299]Specific amino acid residues were identified that, when substituted, yield a mutant ecdysone receptor that exhibits increased activity in response to an ecdysteroid ligand. The effect of an amino acid substitution at amino acid residue 119 of SEQ ID NO: 1 on the activity of the mutated CfEcR-DEF rece...

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Abstract

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic organisms.

Description

[0001]This application claims priority to U.S. provisional application No. 60 / 567,294 filed Apr. 30, 2004 and U.S. provisional application No. 60 / 609,424 filed Sep. 13, 2004.FIELD OF THE INVENTION[0002]This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to novel nuclear receptors comprising a substitution mutation and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.BACKGROUND OF THE INVENTION[0003]Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties. However, the citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.[0004]In the field of genetic engineering, pre...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/00C07H21/04C12N5/00C07K16/00A01K67/00A61K31/16A61K31/47C07D215/16C07K14/435C07K14/705C12N15/62C12P21/06C12Q1/68
CPCA61K31/16A61K31/47C07D215/44C07H21/04C12N15/85C12N15/62Y10S530/858C12N15/63C12N15/635C07K14/43536C07K14/705
Inventor PALLI, SUBBA REDDYKUMAR, MOHAN BASAVARAJU
Owner PRECIGEN INC
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